The solvent chosen for the manufacturing of electrospun polycaprolactone scaffolds influences cell behavior of lung cancer cells

Abstract

The development of a trustworthy in vitro lung cancer model is essential to better understand the illness, find novel biomarkers, and establish new treatments. Polycaprolactone (PCL) electrospun nanofibers are a cost-effective and ECM-like approach for 3D cell culture. However, the solvent used to prepare the polymer solution has a significant impact on the fiber morphology and, consequently, on the cell behavior. Hence, the present study evaluated the effect of the solvent employed in the manufacturing on the physical properties of 15%-PCL electrospun scaffolds and consequently, on cell behavior of NCI-H1975 lung adenocarcinoma cells. Five solvents mixtures (acetic acid, acetic acid-formic acid (3:1, v/v), acetone, chloroform-ethanol (7:3, v/v), and chloroform-dichloromethane (7:3, v/v)) were tested. The highest cell viability ([Formula: see text] = 33.4%) was found for cells cultured on chloroform-ethanol (7:3) PCL scaffolds. Chloroform-dichloromethane (7:3) PCL scaffolds exhibited a roughness that enhanced the quality of electrospun filament, in terms of cell viability. Our findings highlighted the influence of the solvent on fiber morphology and protein adsorption capacity of nanofilaments. Consequently, these features directly affected cell attachment, morphology, and viability.

Figure 1

Scanning electronic microscopy (SEM) images from (A) AA-PCL scaffolds, (B) AA-FA-PCL scaffolds, (C) Ace-PCL scaffolds, (D) Chl-EtOH-PCL scaffolds, and (E) Chl-DCM-PCL scaffolds (scale bar 60 μm).

Figure 2

(A) Storage modulus (E′) and (B) Tan Delta curve obtained by dynamic mechanical analysis (DMA) of PCL scaffolds.

Figure 3

Scanning electronic microscopy (SEM) images from NCI-H1975 cells attached to (A) AA-PCL scaffolds, (B) AA-FA-PCL scaffolds, (C) Ace-PCL scaffolds, (D) Chl-EtOH-PCL scaffolds, and (E) Chl-DCM-PCL scaffolds (scale bar: 30 μm). (F) SEM picture from a NCI-H1975 cell attached to a Chl-DCM-PCL nanofiber due to roughness (scale bar 6 μm). (G) SEM picture from NCI-H1975 cells attached to a monolayer (scale bar 300 μm). (H) SEM picture from NCI-H1975 cells attached to a monolayer (scale bar 30 μm). Representative cells are indicated by arrows.

Figure 4

Pictures of NCI-H1975 cells cultured on monolayer and PCL scaffolds for 3 and 6 days displayed by confocal laser scanning microscope (CLSM) at a magnification of ×200 (scale bars 100 µm). Actin cytoskeleton was stained with rhodamine-phalloidin (red) and nucleus with DAPI (blue).

Figure 5

Cell viability and protein adsorption of NCI-H1975 cells cultured on PCL scaffolds for (A) 3 and (B) 6 days. Cell viability is represented in boxplot form on the left axis. Protein adsorption was normalized by the scaffold weight and is shown as mean with circles on the right axis. Cell viability per fiber of NCI-H1975 cells cultured on PCL scaffolds for (C) 3 and (D) 6 days. (E) Cell viability per fiber (V′). The cell viability was normalized by the porosity and layers of the scaffold to obtain a parameter which represents the cell viability on fibers of each layer.